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1.
Paecilomyces variotii IRI017 was isolated as a formaldehyde-resistant fungus from wastewater containing formaldehyde. The fungus grew in a medium
containing 0.5% formaldehyde and had consumed formaldehyde completely after 5 days. Alcohol oxidase was purified from the
fungus grown on methanol. A 20-fold purification was achieved with a yield of 44%. The molecular mass of the purified enzyme
was estimated to be 73 and 450 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography,
respectively, suggesting that the enzyme consists of six identical subunits. The N-terminal amino acid sequence of the subunit
was TIPDEVDIII. The enzyme showed an absorption spectrum typical of a flavoprotein and had a noncovalently bound flavin different
from FAD, FMN, and riboflavin. The pH optimum of the enzyme activity was pH 6–10. The enzyme was stable in the pH range of
pH 5–10. The enzyme retained full activity after incubation at 50°C for 30 min. The enzyme oxidized not only methanol but
also lower primary alcohols and formaldehyde. The K
m values for methanol, ethanol, and formaldehyde were 1.9, 3.8, and 4.9 mmol l−1, respectively. 相似文献
2.
Keratinase of Doratomyces microsporus 总被引:10,自引:0,他引:10
The fungus Doratomyces microsporus produced an extracellular keratinase during submerged aerobic cultivation in a medium containing a protein inducer for enzyme
synthesis. The keratinase was purified to homogeneity using hydrophobic interaction chromatography followed by gel chromatography.
The molecular weight was estimated to be 33 kDa (from SDS-PAGE analysis) or 30 kDa (by gel chromatography), suggesting a monomeric
structure. The isoelectric point of the enzyme was determined to be around 9. The optimal pH and temperature for keratinolytic
activity were pH 8–9 and 50 °C, respectively. The serine protease inhibitor PMSF totally inhibited the keratinase. The enzyme
was not glycosylated. It was capable of hydrolysing different keratinous materials as well as some non-keratinous proteins.
Hydrolysis of some synthetic substrates, specific for known proteinases, suggested that the keratinase of D. microsporus is close to proteinase K.
Received: 9 July 1999 / Received revision: 13 September 1999 / Accepted: 17 September 1999 相似文献
3.
Jin Zhou Ju Chu Yong-Hong Wang Si-Liang Zhang Ying-Ping Zhuang Zhong-Yi Yuan 《World journal of microbiology & biotechnology》2008,24(6):789-796
An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH4)2SO4 fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed
the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular
weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric
point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5
and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature
stability was up to 45 °C. Metal ions, such as, Mn2+ and K+ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg2+ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu2+ and Ag2+) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K
m of 120 and 330 μM and V
max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively. 相似文献
4.
An extracellular polygalacturonase was isolated from 5-day culture filtrates of Thermoascus aurantiacus CBMAI-756 and purified by gel filtration and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60–65°C.
The apparent K
m with citrus pectin was 1.46 mg/ml and the V
max was 2433.3 μmol/min/mg. The apparent molecular weight of the enzyme was 30 kDa. The enzyme was 100% stable at 50°C for 1 h
and showed a half-life of 10 min at 60°C. Polygalacturonase was stable at pH 5.0–5.5 and maintained 33% of initial activity
at pH 9.0. Metal ions, such as Zn+2, Mn+2, and Hg+2, inhibited 50, 75 and 100% of enzyme activity. The purified polygalacturonase was shown to be an endo/exo-enzyme, releasing
mono, di and tri-galacturonic acids within 10 min of hydrolysis. 相似文献
5.
Hirasawa K Uchimura K Kashiwa M Grant WD Ito S Kobayashi T Horikoshi K 《Antonie van Leeuwenhoek》2006,89(2):211-219
An endoglucanase was purified to homogeneity from an alkaline culture broth of a strain isolated from␣seawater and identified
here as Bacillus agaradhaerens JAM-KU023. The molecular mass was around 38-kDa and the N-terminal 19 amino acids of the purified enzyme exhibited 100% sequence
identity to Cel5A of B. agaradhaerens DSM8721T. The enzyme activity increased around 4-fold by the addition of 0.2–2.0 M NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). KCl,
Na2SO4, NaBr, NaNO3, CH3COONa, LiCl, NH4NO3, and NH4Cl also activated the enzyme up to 2- to 4-fold. The optimal pH and temperature values were pH 7–9.4 and 60 °C with 0.2 M
NaCl, but pH 6.5–7 and 50 °C without NaCl; enzyme activity increased approximately 6-fold at 60 °C with 0.2 M NaCl compared
to that at 50 °C without NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). The thermostability and pH stability of the enzyme were
not affected by NaCl. The enzyme was very stable to several chemical compounds, surfactants and metal ions (except for Fe2+ and Hg2+ ions), regardless whether NaCl was present or not.
* The nucleotide sequence of 16S rRNA of this strain has been submitted to DDBJ, EMBL, and GenBank databases under accession
no. AB211544. 相似文献
6.
The starch-binding domain of Bacillus sp. strain TS-23 α-amylase was introduced into the C-terminal end of Bacillus kaustophilus leucine aminopeptidase (BkLAP) to generate a chimeric enzyme (BkLAPsbd) with raw-starch-binding activity. BkLAPsbd, with an apparent molecular mass of approximately 65 kDa, was overexpressed in Escherichia coli M15 cells and purified to homogeneity by nickel–chelate chromatography. Native PAGE and chromatographic analyses revealed that the purified fusion protein has a hexameric structure. The half-life for BkLAPsbd was 12 min at 70°C, while less than 20% of wild-type enzyme activity retained at the same heating condition. Compared with the wild-type enzyme, the 60% decrease in the catalytic efficiency of BkLAPsbd was due to a 91% increase in K
m value. Starch-binding assays showed that the K
d and B
max values for the fusion enzyme were 2.3 μM and 0.35 μmol/g, respectively. The adsorption of the crude BkLAPsbd onto raw starch was affected by starch concentration, pH, and temperature. The adsorbed enzyme could be eluted from the adsorbent by 2% soluble starch in 20 mM Tris–HCl buffer (pH 8.0). About 49% of BkLAPsbd in the crude extract was recovered through one adsorption–elution cycle with a purification of 11.4-fold. 相似文献
7.
Purification and partial characterization οf a new β-xylosidase from
Humicola grisea var. thermoidea
T. Iembo M.O. Azevedo C. Bloch Jr. E.X.F. Filho 《World journal of microbiology & biotechnology》2006,22(5):475-479
Summary The thermophilic fungus Humicola grisea var. thermoidea produces a mycelium-associated β-xylosidase activity when grown in liquid-state cultures on media containing oat spelt xylan
as the carbon source. The β-xylosidase was purified to apparent homogeneity by gel filtration and anion exchange chromatography.
Its molecular weight was 37 and 50 kDa, as determined by MALDI/TOF mass spectrometry and SDS-PAGE, respectively. The purified
enzyme exhibited maximum activity at 55 °C and pH 6.5. It was also active at pH 8.8, retaining 60% of its activity after 6 h
of incubation at 50 °C. β-xylosidase was strongly inactivated by NBS and slightly activated by DTT and β-mercaptoethanol.
The enzyme was highly specific for PNPX as the substrate. The purified β-xylosidase showed K
m and V
max values of 1.37 mM and 12.98 IU ml−1, respectively. 相似文献
8.
Bacillus pumilis F3-4 utilized feather as a sole source of carbon, nitrogen and sulfur. Supplementation of the feather medium with glucose
or MgSO4 · 7H2O increased keratinolytic protease production (14.6–16.7 U/mg). The synthesis of keratinolytic protease was repressed by an
exogenous nitrogen source. Keratinolytic protease was produced in the absence of feather (9.4 U/mg). Feather degradation resulted
in sulfhydryl group formation (0.8–2.6 μM). B. pumilis F3-4 effectively degraded chicken feather (75%), duck feather (81%) and feather meal (97%), whereas human nails, human hair
and sheep wool under went less degradation (9–15%).
An erratum to this article can be found at 相似文献
9.
Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42 总被引:1,自引:0,他引:1
Kazan D Denizci AA Oner MN Erarslan A 《Journal of industrial microbiology & biotechnology》2005,32(8):335-344
An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease
activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate
by DEAE-cellulose chromatography followed by (NH4)2SO4 precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum
temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca2+ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal
pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses
of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis
by the purified enzyme was 10.59 kcal mol−1 (44.30 kJ mol−1). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h
at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was
not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k
cat value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K
m and k
cat values were estimated at 0.655 μM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21×103 min−1, respectively. 相似文献
10.
The high-molar mass from of β-glucosidase fromAspergillus niger strain NIAB280 was purified to homogeneity with a 46-fold increase in purification by a combination of ammonium sulfate precipitation,
hydrophobic interaction, ion-exchange and gel-filtration chromatography. The native and subunit molar mass was 330 and 110
kDa, respectively. The pH and temperature optima were 4.6–5.3 and 70°C, respectively. TheK
m andk
cat for 4-nitrophenyl β-d-glucopyranoside at 40°C and pH 5 were 1.11 mmol/L and 4000/min, respectively. The enzyme was activated by low and inhibited
by high concentrations of NaCl. Ammonium sulfate inhibited the enzyme. Thermolysin periodically inhibited and activated the
enzyme during the course of reaction and after 150 min of proteinase treatment only 10% activity was lost with concomitant
degradation of the enzyme into ten low-molar-mass active bands. When subjected to 0–9 mol/L transverse urea-gradient-PAGE
for 105 min at 12°C, the nonpurified β-glucosidase showed two major bands which denatured at 4 and 8 mol/L urea, respectively,
with half-lives of 73 min. 相似文献
11.
Stimulation of alkalothermophilic Aspergillus terreusxylanase by low-intensity laser radiation 总被引:1,自引:0,他引:1
In this study, Aspergillus terreus was irradiated by a 7.3 mW He–Ne laser in the presence of crystal violet, toluidine blue O and hematoporphyrin as photosensitizers. Xylanases recovered from non-irradiated and irradiated fungi were purified and characterized. The maximum production of xylanase (42.2 U/ml) was obtained after 5 min of laser irradiation in the absence of the photosensitizer. The irradiation of the sensitized fungus diminished the production of xylanase. On purification using G-100, the specific activity of xylanase recovered from the irradiated fungus was 292 U/mg protein representing a 37-fold purification over the crude extract compared with 95.6 U/mg protein representing the 12.8-fold for the enzyme recovered from the non-irradiated fungus. The enzyme recovered from the irradiated fungus had lower molecular weight as compared with that recovered from the non-irradiated one. Characterization of the purified enzymes revealed that the enzyme recovered from the irradiated fungus was more thermostable and had a wider range of optimum reaction temperature (60–70°C) and pH (4.0–12.0), compared to the non-irradiated one. 相似文献
12.
Mizuho Kusuda Mitsuhiro Ueda Yasuhito Konishi Yoshihito Araki Katsuji Yamanaka Masami Nakazawa Kazutaka Miyatake Takao Terashita 《Mycoscience》2006,47(4):184-189
We investigated extracellular carbohydrase production in the medium of an ectomycorrhizal fungus, Tricholoma matsutake, to reveal its ability to utilize carbohydrates such as starch as a growth substrate and to survey the saprotrophic aspects.
We found β-glucosidase activity in the static culture filtrate of this fungus. The β-glucosidase was purified and characterized.
The purified enzyme was obtained from about 2.1 l static culture filtrate, with 9.0% recovery, and showed a single protein
band on SDS-PAGE. Molecular mass was about 160 kDa. The enzyme was most active around 60°C and pH 5.0, and stable over a pH
of 4.0–8.0 for 30 min at 37°C. The purified enzyme was activated by the presence of Ca2+ and Mn2+ ions (about 2–3 times that of the control). The enzyme readily hydrolyzed oligosaccharides having a β-1,4-glucosidic linkage
such as cellobiose and cellotriose. However, it did not hydrolyze polysaccharides such as avicel and CM-cellulose or oligosaccharides
having an α-glucosidic linkage. Moreover, cellotriose was hydrolyzed by the enzyme for various durations, and the resultant
products were analyzed by TLC. We concluded that the enzyme from T. matsutake seems to be a β-glucosidase because cellotriose with a β-1,4-glucosidic linkage decomposed to glucose during the enzyme reaction. 相似文献
13.
The novel fungus Aspergillus niveus RS2 isolated from rice straw showed relatively high xylanase production after 5 days of fermentation. Of the different xylan-containing
agricultural by-products tested, rice husk was the best substrate; however, maximum xylanase production occurred when the
organism was cultured on purified xylan. Yeast extract was found to be the best nitrogen source for xylanase production, followed
by ammonium sulfate and peptone. The optimum pH for maximum enzyme production was 8 (18.2 U/ml); however, an appreciable level
of activity was obtained at pH 7 (10.9 U/ml). Temperature and pH optima for xylanase were 50°C and 7.0, respectively; however
the enzyme retained considerably high activity under high temperature (12.1 U/ml at 60°C) and high alkaline conditions (17.2 U/ml
at pH 8 and 13.9 U/ml at pH 9). The enzyme was strongly inhibited by Hg2+, while Mn2+ was slight activator. The half-life of the enzyme was 48 min at 50°C. The enzyme was purified by 5.08-fold using carboxymethyl-sephadex
chromatography. Zymogram analysis suggested the presence of a single candidate xylanase in the purified preparation. SDS-PAGE
revealed a molecular weight of approximately 22.5 kDa. The enzyme had K
m and V
max values of 2.5 and 26 μmol/mg per minute, respectively. 相似文献
14.
Park SY Kim JT Kang SG Woo JH Lee JH Choi HT Kim SJ 《Applied microbiology and biotechnology》2007,77(1):107-115
Vibrio sp. GMD509, a marine bacterium isolated from eggs of the sea hare, exhibited lipolytic activity on tributyrin (TBN) plate, and the gene representing lipolytic activity was cloned. As a result,
an open reading frame (ORF) consisting of 1,017 bp (338 aa) was found, and the deduced amino acid sequence of the ORF showed
low similarity (<20%) to α/β hydrolases such as dienelactone hydrolases and esterase/lipase with G–X1–S–X2–G sequence conserved. Phylogenetic analysis suggested that the protein belonged to a new family of esterase/lipase together
with various hypothetical proteins. The enzyme was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme (Vlip509) showed the best hydrolyzing activity toward p-nitrophenyl butyrate (C4) among various p-nitrophenyl esters (C2 to C18), and optimal activity of Vlip509 occurred at 30°C and pH 8.5, respectively. Kinetic parameters toward p-nitrophenyl butyrate were determined as K
m (307 μM), k
cat (5.72 s−1), and k
cat/K
m (18.61 s−1 mM−1). Furthermore, Vlip509 preferentially hydrolyzed the S-enantiomer of racemic ofloxacin ester. Despite its sequence homology to dienelactone hydrolase, Vlip509 showed no dienelactone
hydrolase activity. This study represents the identification of a novel lipolytic enzyme from marine environment. 相似文献
15.
Alfredo E. Pérez-Guzmán Teresa Cruz y Victoria Ramón Cruz-Camarillo Humberto Hernández-Sánchez 《World journal of microbiology & biotechnology》2006,22(9):953-958
Summary An X-prolyl-dipeptidylaminopep tidase (Pep-XP) was purified from the crude intracellular extract of Lactococcus lactis subsp. cremoris NRRL 634 by ion exchange and gel filtration chromatographies. The enzyme was purified 80-fold with a recovery of 6%, and appeared as a single band with a molecular weight of about 80 kDa on polyacrylamide gel electrophoresis with sodium dodecyl sulphate (SDS-PAGE). The peptidase showed its maximal activity on arginyl-proline-p-nitroanilide at pH 7.0 and at a temperature of 45 °C, although there was a good activity of Pep-XP in the pH range of 5.5–7.0 and temperatures between 40 and 50 °C. The Michaelis constant (K
m) and the maximum reaction velocity (V
max) values were 0.92 mM and 7.9 U/mg protein min, respectively. The activity of Pep-XP was completely inhibited by phenylmethanesulphonyl fluoride, an inhibitor of serine peptidases, and metal chelators had little effect on enzyme activity. The purified enzyme hydrolyzed synthetic substrates whose structure is X-Pro-Y like Lys-Pro-pNA, but did not hydrolyse Pro-pNA or azocasein, showing that the enzyme did not have aminopeptidase or endopeptidase activities. 相似文献
16.
P. Tangngamsakul A. Karnchanatat P. Sihanonth P. Sangvanich 《Applied Biochemistry and Microbiology》2011,47(4):412-418
A strain of endophytic fungus EF6 isolated from Thai medicinal plants was found to produce higher levels of extracellular
glucoamylase. This strain produced glucoamylase of culture filtrate when grown on 1% soluble starch. The enzyme was purified
and characterized. Purification steps involved (NH4)2SO4 precipitation, anion exchange, and gel filtration chromatography. Final purification fold was 14.49 and the yield obtained
was 9.15%. The enzyme is monomeric with a molecular mass of 62.2 kDa as estimated by SDS-PAGE, and with a molecular mass of
62.031 kDa estimated by MALDI-TOF spectrometry. The temperature for maximum activity was 60°C. After 30 min for incubation,
glucoamylase was found to be stable lower than 50°C. The activity decrease rapidly when residual activity was retained about
45% at 55°C. The pH optimum of the enzyme activity was 6.0, and it was stable over a pH range of 4.0–7.0 at 50°C. The activity
of glucoamylase was stimulated by Ca2+, Co2+, Mg2+, Mn2+, glycerol, DMSO, DTT and EDTA, and strongly inhibited by Hg2+. Various types of starch were test, soluble starch proved to be the best substrate for digestion process. The enzyme catalyzes
the hydrolysis of soluble starch and maltose as the substrate, the enzyme had K
m values of 2.63, and 1.88 mg/ml and V
max, values of 1.25, and 2.54 U/min/mg protein, and V
max/K
m values of 0.48 and 1.35, respectively. The internal amino acid sequences of endophytic fungus EF6 glucoamylase; RALAN HKQVV
DSFRS have similarity to the sequence of the glucoamylase purified form Thermomyces lanuginosus. From all results indicated that this enzyme is a glucoamylase (1,4-α-D-glucan glucanohydrolase). 相似文献
17.
Extracellular lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NRRL3; production,partial purification and properties 总被引:1,自引:0,他引:1
Four strains of Aspergillus niger were screened for lipase production. Each was cultivated on four different media differing in their contents of mineral components
and sources of carbon and nitrogen. Aspergillus niger NRRL3 produced maximal activity (325U/ml) when grown in 3% peptone, 0.05% MgSO4.7H2O, 0.05% KCl, 0.2% K2HPO4 and 1% olive oil:glucose (0.5:0.5). A. niger NRRL3 lipase was partially purified by ammonium sulphate precipitation. The majority of lipase activity (48%) was located
in fraction IV precipitated at 50–60% of saturation with a 18-fold enzyme purification. The optimal pH of the partial purified
lipase preparation for the hydrolysis of emulsified olive oil was 7.2 and the optimum temperature was 60°C. At 70°C, the enzyme
retained more than 90% of its activity. Enzyme activity was inhibited by Hg2+ and K+, whereas Ca2+ and Mn2+ greatly stimulated its activity. Additionally, the formed lipase was stored for one month without any loss in the activity. 相似文献
18.
The importance of various parameters such as sugarcane juice concentration, pH of the medium, and effects of different solid
supports for maximum secretion of pectin lyase from Penicillium citrinum MTCC 8897 has been studied. The enzyme was purified to homogeneity by Sephadex G-100 and DEAE-cellulose chromatography. The
molecular mass determined by SDS-PAGE was 31 kDa. The K
m and k
cat values were found to be 1 mg/ml and 76 sec−1, respectively. The optimum pH of the purified pectin lyase was 9.0, though it retains activity in the pH 9.0–12.0 range when
exposed for 24 h. The optimum temperature was 50°C, and the pectin lyase was found to be completely stable up to 40°C when
exposed for 1 h. The purified pectin lyase was found efficient in retting of Linum usitatissimum, Cannabis sativa, and Crotalaria juncea.
Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 985–992. 相似文献
19.
Lu L Zhao M Zhang BB Yu SY Bian XJ Wang W Wang Y 《Applied microbiology and biotechnology》2007,74(6):1232-1239
The white rot fungus Pycnoporus sanguineus produced high amount of laccase in the basal liquid medium without induction. Laccase was purified using ultrafiltration,
anion-exchange chromatography, and gel filtration. The molecular weight of the purified laccase was estimated as 61.4 kDa
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme oxidized typical substrates of laccases including
2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate), 2,6-dimethoxyphenol, and syringaldazine. The optimum pH and temperature for the purified
laccase were 3.0 and 65°C, respectively. The enzyme was stable up to 40°C, and high laccase activity was maintained at pH 2.0–5.0.
Sodium azide, l-cysteine, and dithiothreitol strongly inhibited the laccase activity. The purified enzyme efficiently decolorized Remazol
Brilliant Blue R in the absence of added redox mediators. The high production of P. sanguineus laccase as well as its decolorization ability demonstrated its potential applications in dye decolorization. 相似文献
20.
Cathespin B has been purified 750-fold to apparent homogeneity from human and bovine brain cortex using ammonium sulfate fractionation
(30–70%), chromatography on Sephadex G-100, CM-Sephadex C-50, and concanavalin A-Sepharose. Enzyme was assayed fluorometrically
at pH 4.0 with pyridoxyl-hemoglobin in the presence of 1 mM DTT and 1 mM EDTA. Properties of the enzyme from the two sources
proved to be similar. On disc PAGE the purified preparation produced two bands associated with proteinase activity that are
due to existence of two multiple forms of brain cathepsin B with pI 6.1 and 6.8. The enzyme is completely inactivated by thiol-blocking
reagents, leupeptin, E-64, and demands thiol compounds for its ultimate activity. Z-Phe-Ala-CHN2 is a potent inhibitor of the enzyme (K
2nd=1280 M−1s−1) in contrast to Z-Phe-Phe-CHN2 (K
2nd=264 M−1s−1). pH optimum in the reaction of hydrolysis of Pxy-Hb is 4.0–6.0,K
M(app.) =10−5 M. Cathepsin B splits azocasein: pH optimum 5.0–6.0,K
M(app.)=2.2·10−5 M, but inclusion of urea in the incubation medium depresses the azocaseinolytic activity of the enzyme 1.5-fold. It does
not split Lys-NNap, Arg-NMec and is not inhibited by bestatin. The specific activity of brain cathepsin B with Z-Arg-Arg-NNapOMe
at pH 6.0 is 10-fold higher than with Bz-Arg-NNap, Z-Gly-Gly-Arg-NNap is a poor substrate. With Z-Arg-Arg-NMec and Bz-Phe-Val-Arg-NMec
the specific acitivity is 80 and 35%, respectively of that with Z-Phe-Arg-NMec.
Special Issue dedicated to Dr. Eugene Kreps. 相似文献